Circulatory YKL-40 & NLR: Underestimated Prognostic Indicators in Diffuse Glioma

In addition to histopathological parameters, evaluation of associated hematological factors is essential for devising a sensitive prognostic scale in glioma. Increased neutrophil-lymphocyte ratio (NLR), a marker of systemic inflammatory response, has recently been associated with worse outcome in various cancers. Given that glioma progression is characterized by inflammation, aggressive angiogenesis, and invasion, increased levels of systemic human-chitinase-3-like-one protein (YKL-40) have also been linked to poor prognosis. The aim of the present study was to assess the plausible association of YKL-40, NLR, and platelet count with increasing tumor grade, and evaluate their status as independent prognostic factors in terms of overall survival (OS) in treatment naive patients with diffuse glioma. Plasma levels of both biochemical markers in 72 diffuse gliomas, median age 42 years, were compared with 36 controls. Comparison of YKL-40, NLR, and PC with respect to tumor grade was found to be significant for each of the markers (P <0.0001) while an inverse significant correlation was seen for YKL-40 and NLR with OS (r = -0.4619, P <0.0001, and r = -0.5561, P < 0.0001, respectively). NLR was the best performing marker with AUC 0.9417 at 97% specificity. In addition, YKL-40 had a positive correlation with NLR (r = 0.4902, P <0.0001), indicating that expression of both markers was linked to inflammation and tumor progression as they were significantly correlated with tumor grade. Expression of YKL-40 and NLR was independently associated with worse survival (HR 1.0062, P = 0.039, and HR 1.1787, P = 0.0003, respectively), thus establishing their clinical utility as prognosticators for diffuse gliomas.

liomas are aggressive and lethal brain cancers through organized cellular changes that can be measured in blood. The magnitude of activation of this systemic reaction can be measured in terms of circulating white cells, polymorphs, and acute phase proteins (4). In the current decade, blood as a liquid biopsy sample has been recognized as a source of novel biomarkers, and the most practical screening tool. This basis led us to look for circulating inflammatory markers in glioma that could be cost effective and evaluated with minimal invasiveness and higher specificity.
Human chitinase-3-like-protein-1 (CHI3L1, also known as YKL-40), an acute-phase glycoprotein, is elevated in cancers and inflammatory diseases (5) but its clinical application remains restricted. It is secreted by activated macrophages and neutrophils in glioma (6) and is also linked to inflammation (7,8), angiogenesis (9), cell proliferation, and invasion (10). CHI3L1 is considered to promote Th2-type inflammation as well as enhance tissue remodelling and repair (11). In their study, Low et al. have discussed that an increased CHI3L1 expression appears to be an important hallmark of inflammation and cancer has been frequently observed in patients with poor prognosis (12).
Similarly, neutrophils are the most abundant white blood cells, and are the first to be recruited to inflammatory sites. The interaction between neutrophils and lymphocytes in response to inflammation is of significance in tumorgenesis (13). In recent studies, neutrophil-lymophocyte ratio (NLR), a haematological marker of systemic inflammation, has been identified as a crucial prognostic biomarker in different cancers (14,15).  (17).
Likewise, platelet count (PC) is linked to vessel wall integrity and upon activation; these cells release factors which affect angiogenic activity (18). The present assessment was meant to ascertain the plausible association of YKL-40, NLR, and PC with tumor grade, and evaluate their status as independent prognostic markers in terms of overall survival (OS) in therapy naïve glioma patients.

Patients
A total of 72 patients admitted for surgery with an initial radiological diagnosis of suspected glioma were considered for this study. Inclusion

Sample collection and storage
Upon obtaining informed consent, 3 ml of peripheral blood was collected from each presurgery patient and also from healthy volunteers.
Samples were processed at 12 ᵒC, plasma was separated and divided into aliquots, then frozen at -80 °C for further experimentation.

Blood Count
The pre-operative total WBC count along with the percentage of neutrophils and lymphocytes were provided by the pathologist to estimate the absolute neutrophils and lymphocyte counts. NLR and PC were then calculated from this full blood count performed before intervention.

YKL-40 evaluation
Plasma YKL-40 concentrations (ng/mL) were determined by a sandwich ELISA assay using the commercial kit from R&D Systems, USA (Catalog no. DC3L10). The protocol was executed according to the manufacturer's instructions. All samples were analyzed in duplicate. YKL-40 levels were measured as absorbance at 450 nm with the correction wavelength set at 540 nm.

IgE assessment
Serum IgE levels were recorded from the clinical profile of the patients to confirm that they had no infection at the time of blood sampling.
Values were compared within study groups to demarcate that elevated levels of YKL-40 and NLR were due to inflammation on tumor initiation.

Statistical analysis
Kruskal-Wallis test was applied to assess the significance of preoperative levels of each biomarker YKL-40, NLR and PC with tumor grade.

ROC curve analysis
The AUROC analysis provided an optimal cut-off value for YKL-40, NLR, and PC to differentiate between control and glioma patients.

Kaplan-Meier method and log-rank test
In lieu of the above results, comparison of survival curves between control and glioma patients with optimal cut-off value to best predict survival using log-rank (Mante-Cox) test was undertaken.   Higher serum levels of inflammatory marker YKL-40 are associated with poor prognosis as ascertained in studies on cancer (27), and also in inflammatory diseases (28)(29)(30)